CN109307723A - A kind of the UPLC detection method and its discrimination method of cape jasmine and water cape jasmine - Google Patents
A kind of the UPLC detection method and its discrimination method of cape jasmine and water cape jasmine Download PDFInfo
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Abstract
The present invention provides the UPLC detection methods of a kind of cape jasmine and water cape jasmine, it is characterised in that: it is detected for reference substance using Ultra Performance Liquid Chromatography instrument with Geniposide gentiobioside with cape jasmine, Gardenoside, crocin-I, crocin-II.The UPLC detection method of cape jasmine and water cape jasmine of the invention, it can be used for the detection of Geniposide gentiobioside with cape jasmine, Gardenoside, crocin-I, crocin-II, it can also be used in the quality testing of cape jasmine and water cape jasmine, cape jasmine and the multicomponent whole looks of water cape jasmine can be reflected, it simultaneously can be used for the identification of cape jasmine and water cape jasmine, as a result reliable, new method is provided for the control of the quality of cape jasmine and water cape jasmine, is had a good application prospect.
Description
Technical field
The present invention relates to the UPLC detection methods and its discrimination method of a kind of cape jasmine and water cape jasmine.
Background technique
Cape jasmine is the dry mature fruit of madder wort cape jasmine Gardenia jasminoides Ellis, first recorded in
Shennong's Herbal is classified as middle product, has purging intense heat relieving restlessness, reducing fever and causing diuresis, removing pattogenic heat from the blood and toxic material from the body and other effects, is used for Vexed, jaundice
Red, blood strangury and dry pain, blood-head tells nosebleed, red eye, swell pain, pathogenic fire,toxin and furuncles are urinated, are controlled outside for dampening pain.Cape jasmine in commodity market at present
In addition to this, occur being dried to for congener water cape jasmine Gardenia jasminoides Elli, f.logicarpa often
Ripe fruit is medicinal as cape jasmine.And water cape jasmine, primarily as dyestuff and edible pigment, the two is in effect, pharmacological action, toxicity
And clinical application etc. is all not quite similar, therefore cape jasmine and water cape jasmine cannot be used as medicine on an equal basis.
Currently, mainly identified by the difference of the exterior appearances such as character and microscopic features about cape jasmine with water cape jasmine,
It is not high to identify accuracy.
Summary of the invention
To solve the above problems, the present invention provides the UPLC detection methods of a kind of cape jasmine and water cape jasmine, it is characterised in that:
Its operating procedure is as follows:
1) preparation of reference substance solution: Geniposide gentiobioside with cape jasmine, Gardenoside, crocin-I, west safflower are weighed respectively
- II reference substance of glycosides, adds lower alcohol to dissolve, mixed reference substance solution is made；
2) preparation of test solution: taking sample to be tested powder, with rudimentary alcohol extracting, filters to obtain test solution；
3) reference solution being drawn respectively and test solution injecting Ultra Performance Liquid Chromatography instrument, chromatographic condition is as follows:
Chromatographic column: using octadecylsilane chemically bonded silica as filler；Mobile phase: using acetonitrile as mobile phase A, with 0.1%
Phosphoric acid solution is Mobile phase B gradient elution；Gradient elution program is as follows:
Further, step 1) the reference substance Geniposide gentiobioside with cape jasmine, Gardenoside, crocin-I, crocin-
II concentration is respectively solution of every 1ml containing 69.6 μ g, 164.0 μ g, 83.2 μ g, 43.6 μ g.
Further, step 2) is described is extracted as ultrasonic extraction or refluxing extraction.
Further, the time of the extraction is 30min.
Further, the w/v of the step 2) sample to be tested and lower alcohol be 1:400~600, preferably 1:
500。
Further, step 1) and the step 2) lower alcohol are methanol, ethyl alcohol, propyl alcohol；Preferably methanol.
Further, the concentration of the lower alcohol is 30-70%V/V；It is preferred that 50%V/V.
Further, the amount of the step 3) reference solution and test solution injection Ultra Performance Liquid Chromatography instrument is 1 μ
L。
Further, the chromatographic column of the step 3) chromatographic condition is 2.1 × 50mm of Waters BEH C18 column, 1.7 μm；
Column temperature is 30 DEG C, flow velocity 0.3mL/min, wavelength are as follows: 0-10min period 238nm, 10-14min period are 440nm.
The present invention also provides the discrimination methods of a kind of cape jasmine and water cape jasmine, which is characterized in that its operating procedure is as follows:
A, cape jasmine to be checked or water cape jasmine are taken；
B, it is detected by aforementioned UPLC method；
C, fingerprint chromatogram is analyzed.
Further, the finger-print, which is shown as cape jasmine, exclusive characteristic peak in 10-11min；It is shown as water cape jasmine
There is exclusive characteristic peak in 2-4min.
The UPLC detection method of cape jasmine and water cape jasmine of the invention can detect Geniposide gentiobioside with cape jasmine, cape jasmine simultaneously
Glycosides, crocin-I, crocin-II, can be used for the quality testing of cape jasmine and water cape jasmine, can reflect cape jasmine and water cape jasmine is more
The whole looks of ingredient, simultaneously can be used for the identification of cape jasmine and water cape jasmine, be the quality control of cape jasmine and water cape jasmine as a result reliably
System provides new method, has a good application prospect.
Obviously, above content according to the present invention is not being departed from according to the ordinary technical knowledge and customary means of this field
Under the premise of the above-mentioned basic fundamental thought of the present invention, the modification, replacement or change of other diversified forms can also be made.
The specific embodiment of form by the following examples remakes further specifically above content of the invention
It is bright.But the range that this should not be interpreted as to the above-mentioned theme of the present invention is only limitted to example below.It is all to be based on above content of the present invention
The technology realized all belongs to the scope of the present invention.
Detailed description of the invention
Fig. 1 cape jasmine mixes reference substance chromatogram (1. Geniposide gentiobioside with cape jasmine；2. Gardenoside；3. crocin-I；4. west
Carthamic acid-II)
Fig. 2 cape jasmine sample chromatogram figure (4. Geniposide gentiobioside with cape jasmine；5. Gardenoside；7. crocin-I；8. west safflower
Glycosides-II)
Fig. 3 water cape jasmine sample chromatogram figure (5. Geniposide gentiobioside with cape jasmine；6. Gardenoside；8. crocin-I；9. west safflower
Glycosides-II)
The cape jasmine sample chromatogram figure that 50% methanol of Fig. 4 extracts
Fig. 5 Precision Experiment chromatogram
Fig. 6 stability experiment chromatogram
Fig. 7 repeated experiment chromatogram
Specific embodiment
Instrument and reagent
Instrument: U.S. Waters ACQUITY UPLCH-Class System (including quaternary high pressure pump system, column oven,
Autosampler, TUV detector, Empower software chromatographic work station)；BP-121s precision electronic balance (ten a ten thousandths, Zhejiang
River precision instrument Co., Ltd)；(one thousandth, Beijing Sai Duolisi instrument system are limited for BS-200S-WEI precision electronic balance
Company)；KQ-500DB numerical control ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.).
Reagent: acetonitrile, methanol are chromatographically pure, remaining reagent (phosphoric acid etc.) is that analysis is pure, and water is ultrapure water.
Reference substance: Geniposide gentiobioside with cape jasmine reference substance (lot number: wkq16021804), Gardenoside reference substance (lot number:
Wkq16052902) ,-I reference substance of crocin (lot number: wkq16030701) ,-II reference substance of crocin (lot number:
) etc. wkq16030602 it is purchased from Wei Keqi Biotechnology Co., Ltd of Sichuan Province (purity is >=98%).
Sample: cape jasmine derives from Jiangxi Province's Fuzhou Shi, Sichuan Province's Luzhou City；Water cape jasmine derives from Jiangxi Province's Fuzhou Shi；?
It harvests when fruit maturation in October, 2015, is processed by " Chinese Pharmacopoeia " 2015 editions regulations.
The cape jasmine UPLC of the present invention of embodiment 1 detection
1) preparation of reference substance solution: precision weighs Geniposide gentiobioside with cape jasmine, Gardenoside, crocin-I, west respectively
- II reference substance of carthamic acid, adds methanol to dissolve, and mixed reference substance solution is made, and concentration is respectively 69.6 μ gmL-1、164.0μ
g·mL-1、83.2μg·mL-1、43.6μg·mL-1；
2) preparation of test solution: precision weighs cape jasmine drying sample powder (crossing No. 4 sieves) 0.1g and is put into 100mL tool plug
Conical flask in, then accurate that 50% methanol 50mL is added, weighed weight is ultrasonically treated 30min, is cooled to room temperature, then weighed
Weight is supplied bodies lost weight with 50% methanol, is shaken up, with the filtration of 0.22 μm of miillpore filter to get；
3) 1 μ L reference solution and test solution injection Ultra Performance Liquid Chromatography instrument are drawn respectively, and chromatographic condition is as follows:
Chromatographic column: Waters BEH C18 chromatographic column (2.1 × 50mm, 1.7 μm)；Mobile phase: using acetonitrile as mobile phase A,
Using 0.1% phosphoric acid solution as Mobile phase B gradient elution；Detection wavelength 238nm (0-10min), 440nm (10-14min)；Flow velocity
0.3mL/min；30 DEG C of column temperature；Gradient elution program is as follows:
The measurement of UPLC map is carried out to mixing reference substance, cape jasmine sample, records map.Mix reference substance the result is shown in Figure 1, Cape jasmine
Subsample result figure 2.
The water cape jasmine UPLC of the present invention of embodiment 2 detection
1) preparation of reference substance solution: precision weighs Geniposide gentiobioside with cape jasmine, Gardenoside, crocin-I, west respectively
- II reference substance of carthamic acid, adds methanol to dissolve, and mixed reference substance solution is made, and concentration is respectively 69.6 μ gmL-1、164.0μ
g·mL-1、83.2μg·mL-1、43.6μg·mL-1；
2) preparation of test solution: precision weighs water cape jasmine drying sample powder (crossing No. 4 sieves) 0.1g and is put into 100mL tool
In the conical flask of plug, then accurate that 50% methanol 50mL is added, weighed weight is ultrasonically treated 30min, is cooled to room temperature, then claim
Determine weight, supply bodies lost weight with 50% methanol, shake up, with the filtration of 0.22 μm of miillpore filter to get；
3) 1 μ L reference solution and test solution injection Ultra Performance Liquid Chromatography instrument are drawn respectively, and chromatographic condition is as follows:
Chromatographic column: Waters BEH C18 chromatographic column (2.1 × 50mm, 1.7 μm)；Mobile phase: using acetonitrile as mobile phase A,
Using 0.1% phosphoric acid solution as Mobile phase B gradient elution；Detection wavelength 238nm (0-10min), 440nm (10-14min)；Flow velocity
0.3mL/min；30 DEG C of column temperature；Gradient elution program is as follows:
The measurement of UPLC map is carried out to mixing reference substance, water cape jasmine sample, records map.The same Fig. 1 of reference substance result is mixed,
Water cape jasmine sample result is shown in Fig. 3.
The cape jasmine of the present invention of embodiment 3 and water cape jasmine UPLC identify
By cape jasmine chromatogram (Fig. 2) in embodiment 1, water cape jasmine chromatogram (Fig. 3) compares and obtains in embodiment 2: cape jasmine
There are No. 9 peaks of characteristic peak in 10-11min, water cape jasmine has No. 3 peaks of characteristic peak in 2-4min, and No. 3 of No. 9 peaks of cape jasmine and water cape jasmine
Peak is its exclusive characteristic peak respectively, can be used as identification peak.
Beneficial effects of the present invention are illustrated below by way of test example:
1 detection methodologies choice of parameters of test example
1 sample extraction method is investigated
The extraction effect of 5 kinds of pure water, 30% methanol, 50% methanol, 80% methanol, methanol different solvents is investigated.
Precision weighs cape jasmine sample powder 0.1g respectively, is put into stuffed conical flask, and then the different extractions of accurate addition are molten
Agent (pure water, 30% methanol, 50% methanol, 80% methanol, methanol) 50mL, weighed weight are ultrasonically treated 30min, are cooled to room
Temperature is supplied less loss weight with Extraction solvent, is shaken up, with 0.22 μm of filtering with microporous membrane to get test sample.
Find that best using 50% methanol extraction effect, peak shape is best, peak area by the investigation to 5 kinds of Extraction solvents
Maximum is shown in Fig. 4, therefore selects 50% methanol as Extraction solvent.
It is optimized after Extraction solvent determines, and for the ultrasonic extraction time, it is final to determine the preparation of cape jasmine test sample
Method: precision weighs sample powder about 0.1g, is put into the conical flask of tool plug, and then tool is added in the accurate 50% methanol 50mL that measures
Conical flask is filled in, weighed weight is ultrasonically treated 30min, is cooled to room temperature, then weighed weight, supplies weightlessness with 50% methanol, shake
It is even, with 0.22 μm of filtering with microporous membrane to get.
2 chromatographic conditions are investigated
Mobile phase screening: by methanol-water, acetonitrile-water, acetonitrile-methanol-water, -0.1% formic acid water of acetonitrile, acetonitrile -
Multiple mobile phases such as 0.1% phosphoric acid water carry out screening comparison, and the separating effect of -0.1% phosphoric acid water of acetonitrile is preferable as the result is shown, therefore
Select -0.1% phosphoric acid water of acetonitrile.
Elution program is investigated: by chromatogram of the sample on 238nm and 440nm, and on mobile phase variable gradient
There is the change of gradient time of absorption maximum, final optimization pass determines elution program such as the following table 1:
1 chromatographic condition elution program of table
In summary experimental result determines that chromatographic condition is as follows, i.e., Waters BEH C18 chromatographic column (2.1 × 50mm,
1.7μm)；Mobile phase: acetonitrile (A) and 0.1% phosphoric acid water (B), gradient elution program is shown in Table 1；Detection wavelength 238nm (0-
10min), 440nm (10-14min)；Flow velocity 0.3mLmin-1；30 DEG C of column temperature；1 μ L of sampling volume.
The preparation of 3 reference substance solutions
It is appropriate to weigh Geniposide gentiobioside with cape jasmine, Gardenoside, crocin-I ,-II reference substance of crocin for precision respectively,
Add methanol to dissolve, mixed reference substance solution is made, concentration is respectively 69.6 μ gmL-1、164.0μg·mL-1、83.2μg·
mL-1、43.6μg·mL-1。
The preparation of 4 test solutions
Precision weighs drying sample powder (cross No. 4 sieve) about 0.1g and is put into the conical flask of 100mL tool plug, it is then accurate plus
Enter 50% methanol 50mL, weighed weight is ultrasonically treated 30min, is cooled to room temperature, then weighed weight, is supplied with 50% methanol
Bodies lost weight shakes up, with the filtration of 0.22 μm of miillpore filter to get.3 parts of each sample is surveyed by chromatographic condition under " 2 " item
It is fixed.
5 methodological studies
5.1 precision test
It takes with a test solution, according to chromatographic condition continuous sample introduction 6 times under " 2 " item, records 20min map, see figure
5.It is that the relative retention time RSD value at each shared peak is calculated referring to peak for 0.12%-0.6% with Gardenoside peak, opposite peak
The RSD of area is 0.38%-2.49%, shows that instrument precision is good, meets the requirement of finger-print.
5.2 stability test
Take with a test solution, respectively at 0,2,4,8,16, for 24 hours by chromatographic condition sample introduction under " 2 " item, record chromatography
Figure, is shown in Fig. 6.The result shows that the relative retention time RSD value at each shared peak is 0.09%-0.49%, the RSD of relative peak area is
1.01%-2.78% illustrates test solution interior for 24 hours more stable.
5.3 repetitive test
5 parts of same sample are taken, by legal system available test sample solution below " 4 " item, and by chromatographic condition sample introduction under " 2 " item, note
Chromatogram is recorded, sees Fig. 7, the relative retention time RSD value at as a result each shared peak is 0.04%-0.38%, the RSD of relative peak area
For 0.87%-2.94%, shows that this method repeatability is good, can be used for fingerprint map analyzing.
Passing through characteristic spectrum methodological study, it is thus identified that the detection method can be used for the quality testing of cape jasmine and water cape jasmine,
And cape jasmine and water cape jasmine can effectively be identified by this method.
Claims (10)
1. the UPLC detection method of a kind of cape jasmine and water cape jasmine, it is characterised in that: its operating procedure is as follows:
1) preparation of reference substance solution: Geniposide gentiobioside with cape jasmine, Gardenoside, crocin-I, crocin-II are weighed respectively
Reference substance adds lower alcohol to dissolve, and mixed reference substance solution is made；
2) preparation of test solution: taking sample to be tested powder, with rudimentary alcohol extracting, filters to obtain test solution；
3) reference solution being drawn respectively and test solution injecting Ultra Performance Liquid Chromatography instrument, chromatographic condition is as follows:
Chromatographic column: using octadecylsilane chemically bonded silica as filler；Mobile phase: using acetonitrile as mobile phase A, with 0.1% phosphoric acid
Solution is Mobile phase B gradient elution；Gradient elution program is as follows:
2. according to the method described in claim 1, it is characterized by: Geniposide rough gentian in the step 1) mixed reference substance solution
Disaccharide glycosides, Gardenoside, crocin-I, crocin-II concentration be respectively every 1ml containing 69.6 μ g, 164.0 μ g, 83.2 μ g,
43.6μg。
3. according to the method described in claim 1, it is characterized by: step 2) is described to be extracted as ultrasonic extraction or refluxing extraction,
Extraction time is 30min.
4. according to the method described in claim 1, it is characterized by: the bulking value of step 2) sample to be tested and lower alcohol
Than for 1:400~600, preferably 1:500.
5. according to the method described in claim 1, it is characterized by: step 1) and the step 2) lower alcohol be methanol, ethyl alcohol,
Propyl alcohol；Preferably methanol.
6. according to the method described in claim 5, it is characterized by: the concentration of the lower alcohol is 30-70%V/V；It is preferred that
50%V/V.
7. according to the method described in claim 1, it is characterized by: the step 3) reference solution and test solution injection
The amount of Ultra Performance Liquid Chromatography instrument is 1 μ L.
8. according to the method described in claim 1, it is characterized by: the chromatographic column of the step 3) chromatographic condition is Waters
BEH C18 column 2.1 × 50mm, 1.7 μm；Column temperature is 30 DEG C, flow velocity 0.3mL/min, wavelength are as follows: 0-10min period
238nm, the 10-14min period are 440nm.
9. the discrimination method of a kind of cape jasmine and water cape jasmine, which is characterized in that its operating procedure is as follows:
A, cape jasmine to be checked or water cape jasmine are taken；
B, it is detected by UPLC method described in claim 1-8；
C, fingerprint chromatogram is analyzed.
10. discrimination method according to claim 9, which is characterized in that the finger-print is shown as cape jasmine in 10-
11min has exclusive characteristic peak；Being shown as water cape jasmine has exclusive characteristic peak in 2-4min.
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| CN111413429A (en) * | 2020-04-15 | 2020-07-14 | 江苏康缘药业股份有限公司 | Detection method of gardenia intermediate and fingerprint spectrum construction method thereof |
| CN112710765A (en) * | 2019-10-24 | 2021-04-27 | 泰州医药城国科化物生物医药科技有限公司 | Fingerprint detection method of gardenia medicinal material and application thereof |
| CN114324701A (en) * | 2021-12-21 | 2022-04-12 | 中南大学 | Method for rapidly and simultaneously determining content of crocin-1, crocin-2, crocin-3 and crocin-4 |
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Application publication date: 20190205 |